Process for microencapsulation of water soluble substances

ABSTRACT

The present invention relates to a process for the preparation of microparticles, with an extremely high encapsulation rate, comprising a water-soluble substance in a biodegradable polymer, said water-soluble substance and said biodegradable polymer being first incorporated in an organic liquid phase comprising at least one organic non-water miscible solvent. The organic phase is poured into an aqueous liquid phase having a volume which is sufficient to dissolve said organic solvent, said aqueous phase containing a surfactant, the resulting organic-aqueous phase being homogenised in order to perform in one single step the microparticle formation and the organic solvent removal. The thus obtained microparticles show surprisingly good agent retention qualities.

The present invention relates to a process for the preparation ofmicroparticles comprising a water-soluble substance in a biodegradablepolymer.

Many different methods of preparation of microspheres are described inthe literature (Herrmann et al., European Journal of Pharmaceutics andBiopharmaceutics 45 (1998) 75-82). The methods presently used for thepreparation of microspheres from hydrophobic polymers are organic phaseseparation and solvent removal techniques.

The solvent removal techniques can be divided into solvent evaporation,solvent extraction, spray drying and supercritical fluid technology. Insolvent evaporation or solvent extraction techniques, a drug containingorganic polymer solution is emulsified into an aqueous or anotherorganic solution. The drug is dissolved, dispersed or emulsified in theinner organic polymer solution.

These solvent removal techniques for production of microspheres byevaporation or extraction necessitate the step of preparing a stableemulsion of organic droplets before solvent removal. The size andcharacteristics of the final microspheres depend on this step duringwhich a stable emulsion in the presence of the solvent is aprerequisite. The proportions of organic solvent and aqueous phase inthe solvent removal methods are carefully maintained so as to controlthe solvent migration in the aqueous phase. Below a certain ratioorganic solvent/aqueous phase, the formation of droplets is not possibleany more (see H. Sah, “Microencapsulation techniques using ethyl acetateas a dispersed solvent: effects of its extraction rate on thecharacteristics of PLGA microspheres,” Journal of controlled release, 47(3) 1997, 233-245). In some methods, solvent is even added to theaqueous phase in order to saturate it and to prevent the solventmigration during the formation of the primary emulsion.

Several related patents and published applications describe variousaspects of these processes.

EP 0 052 105 B2 (Syntex) describes a microcapsule prepared by the phaseseparation technique using a coacervation agent such as mineral oils andvegetable oils.

EP 0 145 240 B1 (Takeda) discloses a method for encapsulating a watersoluble compound by thickening the inner phase of a W/O emulsion,building a W/O/W and subjecting the emulsion to an “in water drying”process. This method brings different drawbacks such as: the necessityof using a thickening agent to retain the drug, and the multi-stepprocedure including two emulsification steps and the “in water drying”step.

EP 0 190 833 B1 (Takeda) describes a method for encapsulating a watersoluble drug in microcapsules by increasing the viscosity of a primaryW/O emulsion to 150-5,000 cp (by the procedure of increasing the polymerconcentration in the organic phase or by adjusting the temperatures)prior to formation of a second W/O/W emulsion which is then subjected to“in water drying”. The drawbacks of this procedure are the complexity ofthe necessary steps, including formation of two emulsions (W/O andW/O/W) one after the other, and the step of “in-water drying”.

U.S. Pat. No. 5,407,609 (Tice/SRI) describes a microencapsulationprocess for highly water soluble agents. This process involves thedistinct steps of forming a primary O/W emulsion, the external aqueousphase being preferably saturated with polymer solvent. This O/W emulsionis then poured to a large volume of extraction medium in order toextract immediately the solvent. The drawback of this method is that theO/W emulsion is formed in the presence of the organic solvent in a smallvolume. The solvent is subsequently removed by extraction in a largeaqueous volume. The polymeric droplets are prevented to harden in theprimary emulsion, allowing the migration of the drug into the externalphase.

WO 95/11008 (Genentech) describes a method for the encapsulation ofadjuvants into microspheres. The process comprises the three distinctsteps of preparing a primary W/O emulsion, followed by the production ofa W/O/W and finally the hardening of the microspheres by extraction ofthe solvent. As already mentioned above, the drawback of such a methodis the complication due to a multi-step procedure separating dropletproduction from solvent elimination.

EP 0 779 072 A1 (Takeda) describes an “in-water drying” method used forthe removal of solvent after production of a W/O/W or a O/W emulsion. Itis mentioned that the O/W method is preferable for active substancesinsoluble or sparingly soluble in water.

It is an object of the present invention to provide with a new processfor the preparation of microparticles comprising water solublebiologically active substances.

It is still further an object of the present invention to provide with anew process for the preparation of microparticles of high encapsulatingefficiency comprising water soluble biologically active substances.

It is further an object of the present invention to provide with aprocess which allows for a reduction of time of exposure of watersoluble active substances to external water phase in the production ofmicroparticles.

It is further an object of the present invention to avoid the formationof specific emulsions, and the problems they have caused as described inthe prior art in the production of microparticles comprising watersoluble active substances.

To these effects, the present invention relates to a process for thepreparation of microparticles with an extremely high encapsulation ratethanks to the optimal reduction of diffusion for the substance to beencapsulated.

More precisely, the present invention relates to a process for thepreparation of microparticles comprising at least one water-solublesubstance in at least one biodegradable polymer, said water-solublesubstance and said biodegradable polymer being first incorporated in anorganic liquid phase comprising at least one organic non-water misciblesolvent, said organic phase being then poured into an aqueous liquidphase having a volume which is sufficient to dissolve said organicsolvent, said aqueous phase containing a surfactant, the resultingorganic-aqueous phase being homogenised in order to perform in onesingle step the microparticle formation and the organic solvent removal.

The methods available up to now for encapsulating certain compounds andagents, and particularly, water soluble compounds and agents, were notefficient enough for encapsulating water soluble biologically activesubstances due to the high affinity that water soluble biologicallyactive substances have with the aqueous phase.

The present invention has found a solution to this problem by reducingthe time required for encapsulating water soluble biologically activessubstances, and therefore avoiding the problem of formation of theprimary emulsion and solvent removal steps which were far too long andallowed the migration of the water soluble biologically activesubstances into the external aqueous phase.

The microparticle formation and their hardening is performed in onesingle step. After homogenisation, the dispersion is directly filtered.The particles are then harvested and optionally lyophilised.

Using the process of the present invention offers the advantage ofproviding an encapsulation efficiency greater than 50% or 80%.

Furthermore, in the process of the present invention, it has beensurprisingly found that it is possible to obtain microparticles with anextremely high encapsulation efficiency of water soluble activesubstances using a new one step O/W or W/O/W homogenisation process.

One of the specific features in the process of the present invention ischaracterised by the fact that no stable primary emulsion comprisingorganic solvent droplets occurs. Avoiding such a step results in abetter retention of the water-soluble substance.

Furthermore, because of the almost instantaneous lack of organic solventwhen the polymer precipitates and captures the water-soluble substance,no further emulsion stage is observed. The microparticles can thus bedirectly harvested after their formation.

Because the microparticle formation and the solvent removal are donetogether in one single step in this process, the water solublebiologically active substance is quickly kept inside the microparticleswhich have an impermeable wall. Thereby any diffusion external to themicroparticles is at a low level, and the encapsulation rate is veryhigh.

It must also be mentioned that the process of the present inventionavoids the steps of solvent extraction and of solvent evaporation.

The organic solvents used in the process of the present invention arenon-water miscible solvents such as esters (e.g. ethyl acetate, butylacetate), halogenated hydrocarbons (e.g. dichloromethane, chloroform,carbon tetrachloride, chloroethane, dichloroethane, trichloroethane),ethers (e.g. ethyl ether, isopropyl ether), aromatic hydrocarbons (e.g.benzene, toluene, xylene), carbonates (e.g. diethyl carbonate), or thelike. Although these solvents are generally classified by the personskilled in the art as non-water miscible solvent, they are actuallysparingly miscible in water, having a low solubility in water. Forinstance, for ethyl acetate and dichloromethane, the solubility is resp.8.70% and 1.32% (by weight) in water at 20-25° C. (see A. K. DoolittleEd., Properties of individual solvents, in The technology of solventsand plasticizers, chpt. 12. Wiley, N.Y., 1954, pp. 492-742). One of thepreferred solvent is ethyl acetate.

The above-mentioned organic solvents can be used alone or in mixtures oftwo or more different solvents.

The volume of the aqueous phase must be sufficient to dissolve, orextract, the total amount of organic solvent used. If this is not thecase, the microparticles cannot be sufficiently hardened. Those “soft”microparticles may therefore melt among each others during thefiltration process.

Accordingly, the amount of organic solvent is kept as low as possible toget a viscous organic phase and to minimise the necessary volume of theaqueous phase. In all of the following embodiments, the volume of theaqueous phase is chosen to be capable of dissolving at least thecomplete amount of organic solvent.

The maximal value of the ratio solvent/water (w/w) in the presentinvention should therefore preferably be 0.087 and 0.013 for ethylacetate and dichloromethane respectively. In the examples given below,the ratio ethyl acetate/aqueous phase ranges from 0.007 to 0.06. Theencapsulating efficiency improves if the volume of aqueous phaseincreases.

A surfactant is added to the aqueous phase in order to keep theprecipitating biodegradable polymer in fine independent particles. Anideal surfactant gives a viscosity to the aqueous phase that approachesthe viscosity of the organic phase.

An electrolyte may also be optionally added to the aqueous solution tocreate repulsion between the particles and preventing aggregation. As apreferred electrolyte, sodium chloride is used in the aqueous phase andleads to a higher encapsulating efficiency.

The aqueous solution can also be buffered to obtain good pH conditionsfor the drug concerning stability and release.

When a solvent such as ethyl acetate is used, it has been surprisinglyfound that the encapsulation efficiency is increased when using coldsolutions, by optimising the solubility of the solvent in water, byreducing the aqueous solubility of the drug, and by slowing down itsdiffusion. In other words, the present invention achieves the effect offurther reducing the already small amount of diffusion of internalparticle substances to the exterior.

A water-soluble biologically active substance is dispersed as such or asan aqueous solution into one of the above-mentioned non-miscible organicsolvent. In some embodiments of the process, the biologically activesubstance is present in solid state in the organic phase during theentrapment procedure, thus slowing down the solubilisation into theaqueous liquid phase.

The thus obtained liquid organic phase containing the biologicallyactive substance is used to dissolve the biodegradable polymer.

The appropriate biodegradable polymers comprise poly(lactides),poly(glycolides), copolymers thereof or other biodegradable polymerssuch as other aliphatic polymers, polycitric acid, poly-malic acid,polysuccinates, polyfumarates, poly-hydroxybutyrates, polycaprolactones,polycarbonates, polyesteramides, poly-anhydrides, poly(amino acids),polyorthoesters, polycyano-acrylates, polyetheresters, poly(dioxanone)s,copolymers of polyethylene glycol (PEG), polyorthoesters, biodegradablepolyurethanes, polyphosphazenes.

Other biocompatible polymers are polyacrylic acid, poly-methacrylicacid, acrylic acid-methacrylic acid copolymers, dextran stearate,ethylcellulose, acetyl-cellulose, nitrocellulose, etc. These polymersmay be homopolymers or copolymers of two or more monomers, or mixturesof the polymers.

The biologically active substance and the polymer can also beincorporated in separate organic phases. The polymer is dissolved inanother above-mentioned organic non-water miscible solvent. Preferredsolvents include ethyl acetate or dichloromethane. More preferred iswhen the solvent used to dissolve the polymer is the same solvent asthat use for incorporating the biologically active substance. The thusobtained separated organic phases are poured together to form ahomogenous organic phase before addition to the aqueous phase.

If the biologically active substance and/or the biodegradable polymer isnot or is only slightly soluble in one of the above-mentioned solvent,for instance in the preferred solvent ethyl acetate, a sufficient amountof co-solvents such those comprised among the family of benzyl alcohol,DMSO, DMF, ethyl alcohol, methyl alcohol, acetonitrile and the like, mayoptionally be used in that purpose.

A better encapsulating efficiency can be achieved by an appropriatesetting of the physic chemical parameters such as surfactant capacity,viscosity, temperature, ionic strength, pH and buffering potentialduring the homogenisation of the organic inner phase into the aqueousphase. By carefully adjusting the production parameters, theprecipitating polymer can be surprisingly well formed into homogeneouslydispersed particles.

Preferably, the amount of solvent used to dissolve the biodegradablepolymer is kept to a minimum in order to be soluble as quickly aspossible (most preferably at once) in the aqueous phase. If the amountof solvent is high, the amount of aqueous phase has to be too large on apractical point of view.

The concentration of polymer in the organic phase is adjusted to 5-90%(by weight), preferably between about 10 and 50%, depending on thepolymer and solvent used.

In the case that the concentration of polymer in the organic solvent ishigh, the viscosity of this phase, depending on the polymer used, may beincreased.

The viscosity of the polymer solution may be comprised between 1000 and40,000 centipoise (cp) (Brookfield viscosity), more preferably between2,000 and 30,000 cp, even more preferable between 3,000 and 20,000 cp.

Using solvents like ethyl acetate for dissolving the polymer, thesolubility of the solvent in the aqueous phase is increased by loweringthe temperature of both, the organic and the aqueous phases,accelerating the solvent migration and therefore also the encapsulationrate.

In process of the present invention, the temperature of the organicphase ranges between about −10° C. and 30° C., and preferably betweenabout 0° C. and 10° C. For ethyl acetate, the temperature rangespreferably between about 2° C. and 5° C. The temperature of thepolymeric organic phase and the temperature of the aqueous phase are thesame or different and are adjusted in order to increase the solubilityof the solvent in the aqueous phase.

The obtained organic phase for use as the inner polymer and biologicallyactive substance containing phase is added to a aqueous outer phaseunder a homogenisation procedure to give microparticles.

For the homogenisation procedure, a method of creating dispersion isused. This dispersion can be realised for example with any apparatuscapable of shaking, mixing, stirring, homogenising or ultrasonicating.

Different agents influencing the physico-chemical characteristics of theresultant medium may be added. For instance, surfactants, such as forexample an anionic surfactant (e.g. sodium oleate, sodium stearate,sodium lauryl sulfate), a nonionic surfactant (e.g.polyoxyethylene-sorbitant fatty acid ester (Tween 80, Tween 60, productsavailable from Atlas Powder Co, U.S.A.), a polyoxyethylene castor oilderivative (HCO-60, HCO-50, products available from Nikko Chemicals,Japan)), polyvinyl pyrrolidone, polyvinyl alcohol,carboxymethyl-cellulose, lecithin or gelatine.

In specific embodiments of the present invention, a surfactant comprisedamong the family of anionic, non-ionic agents or other agents capable ofreducing the surface tension of the polymeric dispersion can be added.Suitably, therefore, are nonionic surfactants such as Tween (for exampleTween 80), anionic surfactants, nonionic surfactant like polyvinylalcohol or others. These surfactants can, in general, be used alone orin combination with other suitable surfactants. The concentration of thesurfactant is selected in order to disperse and stabilise the polymerparticles, and possibly also to give a viscosity approaching theviscosity of the organic phase.

The preferred concentration of the surfactant in the aqueous phaseranges therefore between about 0.01-50% (by weight), preferably betweenabout 5 and 30%. The viscosity depending on the surfactant used and onits concentration ranges between about 1,000-8,000 cp (Brookfieldviscosity), preferably about 3,000-5,000 cp.

Optionally salts comprised among the family of sodium chloride,potassium chloride, carbonates, phosphates and the like can be added tothe aqueous phase to adjust ionic strength and to create a Zetapotential between the polymer particles, leading to particle repulsion.

Additional buffering agents may be added to the aqueous phase tomaintain a specific pH. So, the internal aqueous phase may besupplemented with a pH regulator for retaining stability or solubilityof the biologically active substance, such as carbonic acid, aceticacid, oxalic acid, citric acid, phosphoric acid, hydrochloric acid,sodium hydroxide, is arginine, lysine or a salt thereof. The pH of theformulations of this invention is generally about 5 to 8, preferablyabout 6.5 to 7.5.

The temperature of the aqueous phase can be adjusted to the temperatureof the inner organic phase. The temperature range is from about −10° C.to 30° C., more preferably between 0° and 10° C. and even morepreferably from between 2° C. and 5° C.

The microparticles of the present invention can be prepared in anydesired size, ranging from 1 μm to about 500 μm, by varying theparameters such as polymer type and concentration in the organic phase,volumes and temperature of the organic and aqueous phase, surfactanttype and concentration, homogenisation time and speed. The mean particlesize of the microparticles ranges generally from 10 to 200 μm, morepreferably from 20 to 200 μm, even more preferably from 30 to 150 μm.

A number of water soluble active substances can be encapsulated by theprocess of the present invention.

Preferably, the encapsulated soluble substance is a peptide, apolypeptide, a protein and their related pharmaceutically acceptablesalts. The salt of peptide is preferably a pharmacologically acceptablesalt. Such salts include salts formed with inorganic acids (e.g.hydrochloric acid, sulphuric acid, nitric acid), organic acids (e.g.carbonic acid, bicarbonic acid, succinic acid, acetic acid, propionicacid, trifluoroacetic acid) etc. More preferably, the salt of peptide isa salt formed with an organic acid (e.g. carbonic acid, bicarbonic acid,succinic acid, acetic acid, propionic acid, trifluoroacetic acid) withgreater preference given to a salt formed with acetic acid. These saltsmay be mono-through tri-salts.

Examples of water soluble active substances which can be encapsulated inthe present invention include, but are not limited to, peptides,polypeptides and proteins such as luteinizing hormone releasing hormone(LHRH) or derivatives of LHRH comprising agonists or antagonists,melanocyte stimulating hormone (MSH), thyrotropin releasing hormone(TRH), thyroid stimulating hormone (TRH), follicule stimulating hormone(FSH), human chorionic gonadotropin (HCG), parathyroid hormone (PTH),human placental lactogen, insulin, somatostatin and derivatives,gastrin, prolactin, adreno-corticotropic hormone (ACTH), growth hormones(GH), growth hormone releasing hormone (GHRH), growth hormone releasingpeptide (GHRP), calcitonin, oxytocin, angiotensin, vasopressin,enkephalins, endorphin, enkephalin, kyotorphine, interferons,interleukins, tumor necrosis factor (TNF), erythropoetin (EPO), colonystimulating factors (G-CSF, GM-CSF, M-CSF), thrombopoietin (TPO),platelet derived growth factor, fibroblast growth factors (FGF), nervegrowth factors (NGF), insulin like growth factors (IGF), amylinpeptides, leptin, RGD peptides, bone morphogenic protein (BMP),substance P, serotonin, GABA, tissue plasminogen activator (TPA),superoxide dismutase (SOD), urokinase, kallikrein, glucagon, human serumalbumin, bovine serum albumin, gamma globulin, immunomodulators (EGF,LPS), blood coagulating factor, lysozyme chloride, polymyxin B,colistin, gramicidin, bacitracin and the like.

A number of other unlimiting example of water soluble substances orparticularly a water soluble form of the following substances can beencapsulated by the process of the present invention.

These substances comprise for instance anticancer drugs such asactinomycin D, bleomycin, busulfan, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine,dacarbazine, daunorubicin, doxorubicin, estramustine, etoposide,floxuridine, fludarabine, fluorouracil, hexamethylmelamine, hydroxyurea,idarubicin, ifosfamide, asparaginase, lomustine, mechlorethamine,melphalan, mercaptopurine, methotrexate, mithramycin, mitomycin C,mitotane, mitozantrone, oxaliplatine, pentostatin, procarbazine,streptozocin, teniposide, thioguanine, thiopeta, vinblastine,vincristine and the like; antibiotics such as tetracyclines,penicillins, sulfisoxazole, ampicillin, cephalosporins, erytromycin,clindamycin, isoniazid, amikacin, chloramphenicol, streptomycin,vancomycin and the like.

Other examples of such substances comprise antivirals such as acyclovir,amantadine, and the like; antipyretics, analgesics and antiinflammatoryagents include acetaminophen, acetylsalicylic acid, methylprodnisolone,ibuprofen diclofenac sodium, indomethacin sodium, flufenamate sodium,pethidine hydrochloride, levorphanol tartrate, morphine hydrochloride,oxymorphone and the like; anesthetics such as lidocaine, xylocaine andthe like; antiulcer agents include metoclopramide, ranitidinehydrochloride, cimetidine hydrochloride, histidine hydrochloride, andthe like anorexics such as dexedrine, phendimetrazine tartrate, and thelike; antitussives such as noscapine hydrochloride, dihydrocodeinephosphate, ephedrine hydrochloride, terbutaline sulfate, isopreterenolhydrochloride, salbutamol sulfate, and the like; antiepileptics such asacetazolamide sodium, ethosuximide, phenytoin sodium, diazepam and thelike; antidepressants such as amoxapine, isocarboxamide, pheneizinesulfate, clomipramine, noxiptilin, imipramine, and the likeanticoagulants such as heparin or warfarin, and the like.

Other unlimiting examples comprise sedatives such as chlorpromazinehydrochloride, scopolamine methylbromide, antihistaminics such asdiphenhydramine hydrochloride, ketotifen fumarate, chlorpheniraminemaleate, methoxy-phenamine hydrochloride and the like.

Other unlimiting examples comprise cardiotonics such as etilefrinehydrochloride, aminophylline and the like; antiasthmatics such asterbutaline sulfate, theophylline, ephedrine, and the like; antifungalssuch as amphotericin B, nystatin, ketoconazole, and the like;antiarrhytmic agents such as propranolol hydrochloride, alprenololhydrochloride, bufetolol hydrochloride, oxyprenolol hydrochloride andthe like; antitubercular agents such as isoniazid, ethambutol, and thelike; hypotensive, diuretic agents such as captopril, ecarazine,mecamylamine hydrochloride, clonidine hydrochloride, bunitrololhydrochloride and the like; hormones such as prednisolone sodiumsulfate, betamethasone sodium phosphate, hexestrol phosphate,dexamethasone sodium sulfate and the like; antigens from bacteria,viruses or cancers, antidiabetics such as glipizide, phenforminhydrochloride, buformin hydrochloride, glymidine sodium, methformin, andthe like; cardiovascular agents such as propanolol hydrochloride,nitroglycerin, hydralazine hydrochloride, prazosin hydrochloride and thelike; diuretics such as spironolactone, furosemide and the like; andenzymes, nucleic acids, plant extracts, antimalarials,psychotherapeutics, hemostatic agents, etc.

The examples that follow are set forth as an aid in understanding thepresent invention, and provide some examples of the many embodimentsthat are potentially available for the present invention. They are notintended to limit the scope of the invention.

EXAMPLE 1

62.5 mg of D-Trp⁶-LHRH acetate (Triptorelin acetate) was added to 20 gof ethyl acetate. The peptide particles were reduced in size with asmall size dispersing apparatus. This peptide suspension was added to 2g of poly(D-L-lactide-co-glycolide) (PLGA) with a ratio of lactide toglycolide of 50:50 and a weight average molecular weight of 45,000. Themixture was stirred at room temperature until the polymer was dissolvedand then placed still at 4° C. The Brookfield viscosity of this solutionwas 15′500 cp. (15.5 Pas).

This organic phase was poured into 675 g of aqueous phase containing 20%(w/w) of Tween 80 and 7 g of sodium chloride and having a temperature of4° C. The homogenisation was performed with a Polytron homogeniserduring 3 minutes.

The microparticles were collected right after the end of thehomogenisation step by filtration. The microparticles were then vacuumdried at room temperature.

The entrapment efficiency was 93%, the mean particle size was 52 μm, andthe residual ethyl acetate was 183 ppm (as determined by GC-MS).

EXAMPLE 2

1250 mg of D-Trp⁶-LHRH acetate (Triptorelin acetate) was added to 200 gof ethyl acetate. The peptide particles were reduced in size with asmall size dispersing apparatus.

40 g of poly(D-L-lactide-co-glycolide) (PLGA) with a ratio of lactide toglycolide of 50:50 and a weight average molecular weight of 45,000 weredissolved in 200 g of ethyl acetate at room temperature.

Both organic phases were poured together and stirred briefly on amagnetic stirrer. The suspension was then let to stand at 4° C. untiluse. This organic phase was poured into 7 kg of aqueous phase containing20% (WAN) of Tween 80 in 67 mM phosphate buffer pH 7.4 and 70 g ofsodium chloride and having a temperature of 4° C. The homogenisation wasperformed during 5 minutes.

The microparticles were collected right after the end of thehomogenisation step by filtration. The microparticles were then vacuumdried at room temperature.

The entrapment efficiency was 76% and the mean particle size was 150 μm.

EXAMPLE 3

125 mg of bovine serum albumin was added to 20 g of ethyl acetate. Thesolid protein particles were reduced in size with a small sizedispersing apparatus This protein suspension was added to 2 g ofpoly(D-L-lactide-co-glycolide) (PLGA) with a ratio of lactide toglycolide of 50:50 and a weight average molecular weight of 45,000. 20 gof additional ethyl acetate were added. The mixture was stirred at roomtemperature until the polymer was dissolved and then placed still at 4°C.

This organic phase was poured into 675 g of aqueous phase containing 20%(W/W) of Tween 80 in 67 mM phosphate buffer pH 7.4 and 7 g of sodiumchloride and having a temperature of 7° C. The homogenisation wasperformed with a Polytron during 3 minutes.

The microparticles were collected right after the end of thehomogenisation step by filtration.

The microparticles were then vacuum dried at room temperature. Theentrapment efficiency was 76% and the mean particle size was 74 μm.

EXAMPLE 4

125 mg of D-Trp⁶-LHRH acetate (Triptorelin acetate) was added to 5 g ofethyl acetate. The peptide particles were reduced in size with a smallsize dispersing apparatus.

4 g of poly(D-L-lactide) polymer were added to this peptide suspension.The mixture was stirred at room temperature until the polymer wasdissolved and then placed still at 8° C.

This organic phase was poured into 675 g of aqueous phase containing 20%(W/W) of Tween 80 in 67 mM phosphate buffer pH 7.4 and 7 g of sodiumchloride and having a temperature of 5° C. The homogenisation wasperformed with a homogeniser during 3 minutes.

The microparticles were collected right after the end of thehomogenisation step by filtration. The microparticles were then vacuumdried at room temperature The entrapment efficiency was 57% and the meanparticle size was 30 μm.

EXAMPLE 5

125 mg of D-Trp⁶-LHRH acetate (Triptorelin acetate) was dissolved in 1.5g of water.

4 g of poly(D-L-lactide-co-glycolide) (PLGA) with a ratio of lactide toglycolide of 50:50 and a weight average molecular weight of 45,000 weredissolved in 40 g of ethyl acetate at room temperature. This organicphase was cooled to 4° C.

The aqueous phase was homogenised into the organic phase. This W/Opreparation was poured into 680 g of aqueous phase containing 20% (w/w)of polyoxyethylene sorbitan fatty acid ester (Tween 80) and 7 g ofsodium chloride and having a temperature of 4° C. The homogenisation wasperformed and the microparticles were collected by filtration. Themicroparticles were then vacuum dried at room temperature.

The entrapment efficiency was 80% and the mean particle size was 60 μm.

EXAMPLE 6

125 mg of vapreotide acetate was dissolved in 2 g of water. 4 g ofpoly(D-L-lactide-co-glycolide) (PLGA) with a ratio of lactide toglycolide of 50:50 and a weight average molecular weight of 45,000 weredissolved in 40 g of ethyl acetate at room temperature. This organicphase was cooled to 4° C.

The aqueous phase was homogenised into the organic phase. This W/Opreparation was poured into 800 g of aqueous phase containing 20% (w/w)of polyoxyethylene sorbitan fatty acid ester (Tween 80) and 8 g ofsodium chloride and having a temperature of 4° C. The homogenisation wasperformed and the microparticles were collected by filtration. Themicroparticles were then vacuum dried at room temperature.

The entrapment efficiency was 76% and the mean particle size was 55 μm.

What is claimed is:
 1. A process for the preparation of microparticlescomprising at least one water-soluble substance in at least onebiodegradable polymer, wherein (a) said water-soluble substance and saidbiodegradable polymer are first incorporated in an organic liquid phasecomprising at least one organic non-water miscible solvent comprisingethyl acetate, (b) said organic phase is poured into an aqueous liquidphase having a volume which is sufficient to dissolve said organicsolvent, said aqueous phase containing a surfactant, and (c) theresulting organic-aqueous phase is homogenised under conditions suchthat microparticle formation and their hardening, by organic solventremoval by extraction thereof into the aqueous liquid phase, areperformed in one single step, without organic solvent evaporation. 2.The process of claim 1 wherein the aqueous phase contains an amount ofan electrolyte.
 3. The process of claim 2 wherein the electrolytecomprises sodium chloride.
 4. The process according to claim 1 whereinthe volumic ratio organic solvent/aqueous phase is comprised between0.007 and 0.06.
 5. The process according to claim 1 wherein thetemperature of the organic phase is comprised between 2° C. and 5° C. 6.The process according to claim 1 wherein the water-soluble substancecomprises a peptide, a polypeptide, a protein or the relatedpharmaceutically acceptable salts thereof.
 7. The process according toclaim 6 wherein the peptide comprises a luteinizing hormone releasinghormone (LHRH) or a derivative thereof.
 8. The process according toclaim 1 wherein the surfactant comprises Tween
 80. 9. Microparticlesobtained according to the process of claim 1.